Human Nod1 Confers Responsiveness to Bacterial Lipopolysaccharides

The immune response to microbial pathogens is initiated by recognition of specific pathogen components by host cells both at the cell surface and in the cytosol. While the response triggered by pathogen products at the surface of immune cells is well characterized, that initiated in the cytosol is poorly understood. Nod1 is a member of a growing family of intracellular proteins with structural homology to apoptosis regulators Apaf-1/Ced-4 and a class of plant disease-resistant gene products. Here we show that bacterial lipopolysaccharides, but not other pathogen components tested, induced TLR4- and MyD88-independent NF-κB activation in human embryonic kidney 293T cells expressing trace amounts of Nod1. Nod2, another Nod family member, also conferred responsiveness to bacterial components but with a response pattern different from that observed with Nod1. As it was reported for plant disease-resistant R proteins, the leucine-rich repeats of Nod1 and Nod2 were required for lipopolysaccharide-induced NF-κB activation. A lipopolysaccharide binding activity could be specifically coimmunopurified with Nod1 from cytosolic extracts. These observations suggest that Nod1 and Nod2 are mammalian counterparts of plant disease-resistant gene products that may function as cytosolic receptors for pathogen components derived from invading bacteria. The immune response to microbial pathogens is initiated by recognition of specific pathogen components by host cells both at the cell surface and in the cytosol. While the response triggered by pathogen products at the surface of immune cells is well characterized, that initiated in the cytosol is poorly understood. Nod1 is a member of a growing family of intracellular proteins with structural homology to apoptosis regulators Apaf-1/Ced-4 and a class of plant disease-resistant gene products. Here we show that bacterial lipopolysaccharides, but not other pathogen components tested, induced TLR4- and MyD88-independent NF-κB activation in human embryonic kidney 293T cells expressing trace amounts of Nod1. Nod2, another Nod family member, also conferred responsiveness to bacterial components but with a response pattern different from that observed with Nod1. As it was reported for plant disease-resistant R proteins, the leucine-rich repeats of Nod1 and Nod2 were required for lipopolysaccharide-induced NF-κB activation. A lipopolysaccharide binding activity could be specifically coimmunopurified with Nod1 from cytosolic extracts. These observations suggest that Nod1 and Nod2 are mammalian counterparts of plant disease-resistant gene products that may function as cytosolic receptors for pathogen components derived from invading bacteria. Toll-like receptor antibody interleukin IL1 receptor lipopolysaccharide leucine-rich repeat(s) lipoteichoic acid nucleotide binding domain nuclear factor-κB, PGN, peptidoglycan disease-resistant synthetic bacterial lipoprotein hemagglutinin human embryonic kidney tumor necrosis factor The innate immune system regulates the immediate response to microbial pathogens in multiple organisms including humans. The innate immune response is initiated by recognition of specific pathogen components by host immune cells. Mammalian cells have cell surface receptors and intracellular mechanisms that initiate the defense response against microbial pathogens (1Aderem A. Ulevitch R.J. Nature. 2000; 406: 785-787Crossref Scopus (2632) Google Scholar, 2Philpott D.J. Yamaoka S. Israel A. Sansonetti P.J. J. Immunol. 2000; 165: 903-914Crossref PubMed Scopus (216) Google Scholar). Toll-like receptors (TLRs)1 comprise a family of cell surface receptors that are related to the DrosophilaToll protein, a molecule involved in defense against fungal infection in the fly (1Aderem A. Ulevitch R.J. Nature. 2000; 406: 785-787Crossref Scopus (2632) Google Scholar). Ten mammalian TLRs have been identified (1Aderem A. Ulevitch R.J. Nature. 2000; 406: 785-787Crossref Scopus (2632) Google Scholar). Two members of the family, TLR2 and TLR4, have been better characterized and shown to mediate the response to multiple bacterial cell wall components including lipopolysaccharide (LPS), lipopeptides, peptidoglycans (PGN), and lipoteichoic acid (LTA) (3Yang R.B. Mark M.R. Gray A. Huang A. Xie M.H. Zhang M. Goddard A. Wood W.I. Gurney A.L. Godowski P.J. Nature. 1998; 395: 284-288Crossref PubMed Scopus (1102) Google Scholar, 4Poltorak A. He X. Smirnova I. Liu M.Y. Huffel C.V. Du X. Birdwell D. Alejos E. Silva M. Galanos C. Freudenberg M. Ricciardi-Castagnoli P. Layton B. Beutler B. Science. 1998; 282: 2085-2088Crossref PubMed Scopus (6478) Google Scholar, 5Aliprantis A.O. Yang R.B. Mark M.R. Suggett S. Devaux B. Radolf J.D. Klimpel G.R. Godowski P. Zychlinsky A. Science. 1999; 285: 736-739Crossref PubMed Scopus (1278) Google Scholar, 6Chow J.C. Young D.W. Golenbock D.T. Christ W.J. Gusovsky F. J. Biol. Chem. 1999; 274: 10689-10692Abstract Full Text Full Text PDF PubMed Scopus (1626) Google Scholar, 7Schwandner R. Dziarski R. Wesche H. Rothe M. Kirschning C.J. J. Biol. Chem. 1999; 274: 17406-17409Abstract Full Text Full Text PDF PubMed Scopus (1438) Google Scholar). Mammalian TLRs have multiple leucine-rich repeats in the ectodomain and an intracellular Toll-IL1 receptor domain that mediates a signaling cascade to the nucleus (1Aderem A. Ulevitch R.J. Nature. 2000; 406: 785-787Crossref Scopus (2632) Google Scholar). Stimulation of TLR2 and TLR4 leads to the recruitment of the adaptor molecule MyD88 and the serine kinase IL1R-associated kinase, two signaling components that, together with TRAF6, mediate activation of NF-κB (1Aderem A. Ulevitch R.J. Nature. 2000; 406: 785-787Crossref Scopus (2632) Google Scholar). Plants have several classes of genes that regulate the defense against invading pathogens. An important class of these molecules is termed disease-resistant (R) proteins, and members include both membrane-bound and cytosolic proteins. These are essential for the defense against multiple pathogens including bacteria, fungi, and viruses (8Dixon M.S. Golstein C. Thomas C.M. van Der Biezen E.A. Jones J.D. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8807-8814Crossref PubMed Scopus (128) Google Scholar). The cytosolic type of R proteins, which include the tobacco N gene product and up to 200 gene products inArabinopsis thaliana, are comprised of an N-terminal Toll-IL1 receptor or leucine zipper effector domain, a centrally located nucleotide binding domain (NBD), and C-terminal leucine-rich repeats (LRRs). The LRRs of cytosolic R proteins are highly diverse and appear to be involved in the recognition of a wide array of microbial components (8Dixon M.S. Golstein C. Thomas C.M. van Der Biezen E.A. Jones J.D. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8807-8814Crossref PubMed Scopus (128) Google Scholar). This class of disease-resistant proteins mediates the hypersensitive response in plants that includes metabolic alterations and localized cell death at the site of pathogen invasion (8Dixon M.S. Golstein C. Thomas C.M. van Der Biezen E.A. Jones J.D. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8807-8814Crossref PubMed Scopus (128) Google Scholar). The cytosolic R proteins of plants have remarkable structural homology to Nod1/Card 4, a recently described protein related to the apoptosis regulator Apaf-1 (9Zou H. Henzel W.J. Liu X. Lutschg A. Wang X. Cell. 1997; 90: 405-413Abstract Full Text Full Text PDF PubMed Scopus (2746) Google Scholar, 10Bertin J. Nir W.J. Fischer C.M. Tayber O.V. Errada P.R. Grant J.R. Keilty J.J. Gosselin M.L. Robison K.E. Wong G.H. Glucksmann M.A. DiStefano P.S. J. Biol. Chem. 1999; 274: 12955-12958Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar, 11Inohara N. Koseki T. del Peso L. Hu Y. Yee C. Chen S. Carrio R. Merino J. Liu D. Ni J. Núñez G. J. Biol. Chem. 1999; 274: 14560-14568Abstract Full Text Full Text PDF PubMed Scopus (630) Google Scholar). Like plant R proteins, Nod1 is comprised of an N-terminal effector domain, a centrally located NBD, and multiple LRRs at the C terminus (10Bertin J. Nir W.J. Fischer C.M. Tayber O.V. Errada P.R. Grant J.R. Keilty J.J. Gosselin M.L. Robison K.E. Wong G.H. Glucksmann M.A. DiStefano P.S. J. Biol. Chem. 1999; 274: 12955-12958Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar, 11Inohara N. Koseki T. del Peso L. Hu Y. Yee C. Chen S. Carrio R. Merino J. Liu D. Ni J. Núñez G. J. Biol. Chem. 1999; 274: 14560-14568Abstract Full Text Full Text PDF PubMed Scopus (630) Google Scholar). Nod1 induces NF-κB activation, which is mediated through the association of its N-terminal caspase recruitment domain with that of RICK, a protein kinase that also activates NF-κB (10Bertin J. Nir W.J. Fischer C.M. Tayber O.V. Errada P.R. Grant J.R. Keilty J.J. Gosselin M.L. Robison K.E. Wong G.H. Glucksmann M.A. DiStefano P.S. J. Biol. Chem. 1999; 274: 12955-12958Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar, 11Inohara N. Koseki T. del Peso L. Hu Y. Yee C. Chen S. Carrio R. Merino J. Liu D. Ni J. Núñez G. J. Biol. Chem. 1999; 274: 14560-14568Abstract Full Text Full Text PDF PubMed Scopus (630) Google Scholar, 12Inohara N. del Peso L. Koseki T. Chen S. Núñez G. J. Biol. Chem. 1998; 273: 12296-12300Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar, 13McCarthy J.V. Ni J. Dixit V.M. J. Biol. Chem. 1998; 273: 16968-16975Abstract Full Text Full Text PDF PubMed Scopus (370) Google Scholar, 14Thome M. Hofmann K. Burns K. Martinon F. Bodmer J.L. Mattmann C. Tschopp J. Curr. Biol. 1998; 8: 885-888Abstract Full Text Full Text PDF PubMed Google Scholar, 15Inohara N. Koseki T. Lin J. del Peso L. Lucas P.C. Chen F.F. Ogura Y. Nuñez G. J. Biol. Chem. 2000; 275: 27823-27831Abstract Full Text Full Text PDF PubMed Scopus (454) Google Scholar). However, the trigger molecule(s) that activates Nod1 to mediate NF-κB activation remains unknown. Given the structural homology between Nod1 and intracellular LRR-NBD-containing plant R proteins, we performed experiments to determine whether Nod1 might regulate the cellular response to microbial components. Here we report that expression of Nod1 confers responsiveness to LPS and provide evidence that Nod1 exhibits LPS binding activity. Therefore, we propose that Nod1 and Nod2, another Nod family member recently identified, are cytosolic receptors for pathogen components and functional counterparts of intracellular plant R gene products. LPS from various sources in this study were obtained from Sigma. PGN from Staphylococcus aureuswas obtained from Fluka-Chemie (Buchs, Germany). Mannan fromCandida albicans 20A was a gift of P. Lehmann (Medical College of Ohio). Pam3CysSerLys4, a synthetic bacterial lipoprotein analogue (sBLP), was a gift of A. Zychlinsky (New York University School of Medicine). The expression plasmids pcDNA3-Nod1-FLAG, pcDNA3-Nod1(1–648)-FLAG, pcDNA3-Nod2, pcDNA3-Nod2(1–744)-FLAG, pRK7-FLAG-IKKβ, pcDNA3-FLAG-IKKi, pcDNA3-CIPER-FLAG, pCMV-IL1R, pCMV-TLR4-FLAG, pcDNA3-FLAG-RIP, pcDNA3-MyD88 DN (amino acids 1–109), pcDNA3-CD14, pCMV-MD2-FLAG, and pcDNA3-β-galactosidase have been described previously (11Inohara N. Koseki T. del Peso L. Hu Y. Yee C. Chen S. Carrio R. Merino J. Liu D. Ni J. Núñez G. J. Biol. Chem. 1999; 274: 14560-14568Abstract Full Text Full Text PDF PubMed Scopus (630) Google Scholar, 12Inohara N. del Peso L. Koseki T. Chen S. Núñez G. J. Biol. Chem. 1998; 273: 12296-12300Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar, 15Inohara N. Koseki T. Lin J. del Peso L. Lucas P.C. Chen F.F. Ogura Y. Nuñez G. J. Biol. Chem. 2000; 275: 27823-27831Abstract Full Text Full Text PDF PubMed Scopus (454) Google Scholar,17Shimada T. Kawai T. Takeda K. Matsumoto M. Inoue J. Tatsumi Y. Kanamaru A. Akira S. Int. Immunol. 1999; 11: 1357-1362Crossref PubMed Scopus (312) Google Scholar, 18Huang J. Gao X. Li S. Cao Z. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12829-12832Crossref PubMed Scopus (195) Google Scholar, 19Medzhitov R. Preston-Hurlburt P. Kopp E. Stadlen A. Chen C. Ghosh S. Janeway Jr., C.A. Mol. Cell. 1998; 2: 253-258Abstract Full Text Full Text PDF PubMed Scopus (1311) Google Scholar, 20Hsu H. Huang J. Shu H.B. Baichwal V. Goeddel D.V. Immunity. 1996; 4: 387-396Abstract Full Text Full Text PDF PubMed Scopus (983) Google Scholar). 2Ogura, Y., Inohara, N., Benito, A., Chen, F. F., and Nuñez, G. (2001) J. Biol. Chem., in press. To construct the expression plasmid producing C-terminally HA-tagged mature IL1β, pcDNA3-mIL1β-HA, the mature region of mouse IL1β, was amplified by polymerase chain reaction and inserted into pcDNA3-HA-pro, which contains the signal sequence of protrypsin and the HA tag. HEK293T cells were transfected, and NF-κB activation assays were performed as described (15Inohara N. Koseki T. Lin J. del Peso L. Lucas P.C. Chen F.F. Ogura Y. Nuñez G. J. Biol. Chem. 2000; 275: 27823-27831Abstract Full Text Full Text PDF PubMed Scopus (454) Google Scholar). 1 × 108HEK293T cells were transfected with expression plasmids indicated in figure legends as described (15Inohara N. Koseki T. Lin J. del Peso L. Lucas P.C. Chen F.F. Ogura Y. Nuñez G. J. Biol. Chem. 2000; 275: 27823-27831Abstract Full Text Full Text PDF PubMed Scopus (454) Google Scholar). 24 h post-transfection, S100 fractions were prepared from transfected cells as described (4Poltorak A. He X. Smirnova I. Liu M.Y. Huffel C.V. Du X. Birdwell D. Alejos E. Silva M. Galanos C. Freudenberg M. Ricciardi-Castagnoli P. Layton B. Beutler B. Science. 1998; 282: 2085-2088Crossref PubMed Scopus (6478) Google Scholar). For Fig. 3 A, S100 lysate containing 5 mg of protein was incubated with 300 ng of [3H]LPS (1 × 105 Bq, 347 Bq/ng; List Biological Laboratories, Campbell, CA) from Escherichia coli K12 LCD25, 6 μg of anti-FLAG M2 antibody (Sigma), 10 μl of protein A-Sepharose, and 10 μl of protein G-Sepharose at 4 °C for 2 h. Proteins bound to the matrix were washed 5 times with 1 ml of Buffer A. The bound radioactivity was measured using a Beckman LS5000LD liquid scintillation counter. For Fig. 3 B, proteins were immunopurified first from 20 mg of S100 lysate as described above and then incubated with 300 ng of [3H]LPS in the presence of 10 mg of bovine serum albumin Fraction V (Sigma) at 4 °C for 2 h. After 5 washes with 1 ml of Buffer A, the bound radioactivity was measured. To monitor protein expression, proteins in 50 μg of S100 lysate were detected by immunoblotting with anti-FLAG Ab. To study a role of Nod1 in the cellular response to microbial components, HEK293T cells were transiently cotransfected with a Nod1 expression plasmid or control plasmid and an NF-κB reporter construct in the presence of bacterial or fungal products. No significant induction of reporter gene activity was observed when the cells transfected with control plasmid were exposed to LPS, PGN, LTA, sBLP, or mannan (Fig. 1 A). These results are in agreement with previous observations in HEK293 cells (3Yang R.B. Mark M.R. Gray A. Huang A. Xie M.H. Zhang M. Goddard A. Wood W.I. Gurney A.L. Godowski P.J. Nature. 1998; 395: 284-288Crossref PubMed Scopus (1102) Google Scholar,5Aliprantis A.O. Yang R.B. Mark M.R. Suggett S. Devaux B. Radolf J.D. Klimpel G.R. Godowski P. Zychlinsky A. Science. 1999; 285: 736-739Crossref PubMed Scopus (1278) Google Scholar, 6Chow J.C. Young D.W. Golenbock D.T. Christ W.J. Gusovsky F. J. Biol. Chem. 1999; 274: 10689-10692Abstract Full Text Full Text PDF PubMed Scopus (1626) Google Scholar, 7Schwandner R. Dziarski R. Wesche H. Rothe M. Kirschning C.J. J. Biol. Chem. 1999; 274: 17406-17409Abstract Full Text Full Text PDF PubMed Scopus (1438) Google Scholar). Because overexpression of Nod1 induces NF-κB activation (10Bertin J. Nir W.J. Fischer C.M. Tayber O.V. Errada P.R. Grant J.R. Keilty J.J. Gosselin M.L. Robison K.E. Wong G.H. Glucksmann M.A. DiStefano P.S. J. Biol. Chem. 1999; 274: 12955-12958Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar,11Inohara N. Koseki T. del Peso L. Hu Y. Yee C. Chen S. Carrio R. Merino J. Liu D. Ni J. Núñez G. J. Biol. Chem. 1999; 274: 14560-14568Abstract Full Text Full Text PDF PubMed Scopus (630) Google Scholar), we transfected HEK293T cells with 0.3 ng of Nod1 and measured NF-κB activation after incubation with various pathogen components. LPS, but not the other microbial products tested, induced significant NF-κB activation (about 12-fold) in cells expressing trace amounts of Nod1 (Fig. 1 A). To demonstrate that NF-κB activation by LPS is specific for cells expressing Nod1, HEK293T cells were transfected with expression plasmids producing IL1R and its ligand IL1β or RIP, a mediator of the TNFα signaling pathway (18Huang J. Gao X. Li S. Cao Z. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12829-12832Crossref PubMed Scopus (195) Google Scholar, 20Hsu H. Huang J. Shu H.B. Baichwal V. Goeddel D.V. Immunity. 1996; 4: 387-396Abstract Full Text Full Text PDF PubMed Scopus (983) Google Scholar). As expected, stimulation of the IL1R and expression of RIP induced NF-κB activation in the absence of LPS (Fig. 1 B). Significantly, LPS did not enhance NF-κB activation induced by IL1R stimulation or RIP (Fig. 1 B). Plant disease-resistant proteins have C-terminal LRRs that are critical for pathogen-specific responses (8Dixon M.S. Golstein C. Thomas C.M. van Der Biezen E.A. Jones J.D. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8807-8814Crossref PubMed Scopus (128) Google Scholar). Alterations in their LRRs result in unresponsiveness to particular pathogens (8Dixon M.S. Golstein C. Thomas C.M. van Der Biezen E.A. Jones J.D. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8807-8814Crossref PubMed Scopus (128) Google Scholar), suggesting that the LRRs of Nod1 might be also required for the response to LPS. To test this hypothesis, HEK293T cells were transfected with plasmids expressing wild-type or truncated Nod1 mutant lacking the LRRs (Nod1ΔLRR) and treated with LPS. Expression of Nod1ΔLRR induced higher NF-κB activation than wild-type Nod1 in the absence of LPS, as previously reported (11Inohara N. Koseki T. del Peso L. Hu Y. Yee C. Chen S. Carrio R. Merino J. Liu D. Ni J. Núñez G. J. Biol. Chem. 1999; 274: 14560-14568Abstract Full Text Full Text PDF PubMed Scopus (630) Google Scholar). Significantly, LPS did not enhance NF-κB activation induced by Nod1ΔLRR (Fig.2). Thus, the LRRs are essential for Nod1 to respond to LPS. Several studies have provided conclusive evidence that TLR4 is a cell surface receptor for LPS (1Aderem A. Ulevitch R.J. Nature. 2000; 406: 785-787Crossref Scopus (2632) Google Scholar, 4Poltorak A. He X. Smirnova I. Liu M.Y. Huffel C.V. Du X. Birdwell D. Alejos E. Silva M. Galanos C. Freudenberg M. Ricciardi-Castagnoli P. Layton B. Beutler B. Science. 1998; 282: 2085-2088Crossref PubMed Scopus (6478) Google Scholar, 6Chow J.C. Young D.W. Golenbock D.T. Christ W.J. Gusovsky F. J. Biol. Chem. 1999; 274: 10689-10692Abstract Full Text Full Text PDF PubMed Scopus (1626) Google Scholar, 21Takeuchi O. Hoshino K. Kawai T. Sanjo H. Takada H. Ogawa T. Takeda K. Akira S. Immunity. 1999; 4: 443-451Abstract Full Text Full Text PDF Scopus (2800) Google Scholar). Therefore, it is possible that expression of Nod1 confers LPS responsiveness through TLR4. To test this possibility, HEK293T cells were cotransfected with a TLR4 expression plasmid, and NF-κB activity was measured in the presence and absence of LPS. Expression of TLR4 alone did not induce NF-κB activation in the presence of LPS, which is consistent with recent reports that additional cell surface molecules such as MD2 and CD14 are required for TLR4-mediated LPS responses in cells (6Chow J.C. Young D.W. Golenbock D.T. Christ W.J. Gusovsky F. J. Biol. Chem. 1999; 274: 10689-10692Abstract Full Text Full Text PDF PubMed Scopus (1626) Google Scholar, 21Takeuchi O. Hoshino K. Kawai T. Sanjo H. Takada H. Ogawa T. Takeda K. Akira S. Immunity. 1999; 4: 443-451Abstract Full Text Full Text PDF Scopus (2800) Google Scholar). In accord with the latter, cotransfection of TLR4, CD14, and MD2 expression plasmids induced 8-fold activation of NF-κB (see legend for Fig.1 C). To further verify that Nod1 confers LPS responsiveness independently of TLRs, a dominant negative mutant of MyD88, a common signaling molecule of IL1, and Toll-related receptors including TLR4 were coexpressed with Nod1 or TLR4, CD14, and MD2 as a control, and transfected cells were stimulated with LPS. Coexpression of the MyD88 mutant suppressed NF-κB activation induced by both TLR4 and IL1R stimulation, but it did not affect LPS-mediated NF-κB activation induced by Nod1 (Fig. 1 C). Furthermore, expression of a dominant negative mutant of TRAF6, a signaling molecule of TLR signaling pathways (22Shimazu R. Akashi S. Ogata H. Nagai Y. Fukudome K. Miyake K. Kimoto M.J. Exp. Med. 1999; 189: 1777-1782Crossref PubMed Scopus (1760) Google Scholar), did not block NF-κB activation induced by Nod1 but inhibited TLR4-mediated NF-κB activation (11Inohara N. Koseki T. del Peso L. Hu Y. Yee C. Chen S. Carrio R. Merino J. Liu D. Ni J. Núñez G. J. Biol. Chem. 1999; 274: 14560-14568Abstract Full Text Full Text PDF PubMed Scopus (630) Google Scholar). These results indicate that NF-κB activation in Nod1-expressing cells induced by LPS is not mediated by the TLR4 signaling pathway. Consistent with this notion are recent observations showing that the Nod1 signaling pathway leading to NF-κB activation is distinct to that of TLRs. Nod1 activates NF-κB through its association with RICK, a protein kinase that directly interacts with IKKγ/NEMO, the regulatory subunit of the IκB kinase complex (15Inohara N. Koseki T. Lin J. del Peso L. Lucas P.C. Chen F.F. Ogura Y. Nuñez G. J. Biol. Chem. 2000; 275: 27823-27831Abstract Full Text Full Text PDF PubMed Scopus (454) Google Scholar). LPS from different Gram-negative bacteria have diverse structures (23Rietschel E.T. Brade H. Holst O. Brade L. Müller-Loennies S. Mamat U. Zähringer U. Beckmann F. Seydel U. Brandenburg K. Ulmer A.J. Mattern T. Heine H. Schletter S. Hauschildt S. Loppnew H. Schönbeck U. Flad H.-D. Schade U.F. DiPadova F. Kusumoto S. Schumann R.R. Curr. Top. Microbiol. Immunol. 1997; 216: 39-81Google Scholar). To determine whether Nod1 confers responsiveness to LPS from several bacterial sources, Nod1-expressing cells were stimulated with LPS from six pathogenic bacteria or TNFα as a positive control. All LPS preparations induced NF-κB activation in Nod1-expressing cells, but different sources of LPS differed in their ability to enhance Nod1-mediated NF-κB activation (Fig. 2). As it was found with LPS from E. coli 055:B5 (Fig. 1 B), none of the LPS preparations induced significant NF-κB activation in cells expressing a Nod1 mutant lacking the LRRs (Fig. 2). Plants have numerous disease-resistant R genes, and mammalian, as well as insect, cells have multiple TLR family members to respond to different pathogens (8Dixon M.S. Golstein C. Thomas C.M. van Der Biezen E.A. Jones J.D. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8807-8814Crossref PubMed Scopus (128) Google Scholar). Notably, we identified human Nod2, another Nod1-like protein that is homologous to Nod1 (34% amino acid identity) and is comprised of N-terminal caspase recruitment domains, NBD and LRRs.2 The presence of multiple Nod family members suggests that Nod1 and Nod2 may have different specificities for pathogen components. To test this, HEK293T cells were cotransfected with plasmids expressing wild-type or mutant Nod2 lacking the LRRs. As it was observed with Nod1, all LPS preparations including those from invasive bacteria such as Salmonella and Shigellastimulated NF-κB activation in cells expressing wild-type Nod2 but not mutant Nod2 (Fig. 2). Notably, LPS from Sarratia marcescens and Salmonella typhimurium was more effective in inducing NF-κB activation in cells expressing Nod2 than Nod1 (Fig. 2). Furthermore, PGN preparation from S. aureusstimulated NF-κB activation in cells expressing Nod2 but not Nod1 (Fig. 2). The molecular basis for the differential response of Nod1 and Nod2 to both LPS and PGN is unclear. Further biochemical analyses and structure determination of the LPS moiety recognized by Nod1 and Nod2 are required to understand the differential response of Nod proteins to bacterial components. Apaf-1, a Nod1-like molecule that plays a central role in apoptosis, mediates responsiveness to cytochrome c leaked from mitochondria (9Zou H. Henzel W.J. Liu X. Lutschg A. Wang X. Cell. 1997; 90: 405-413Abstract Full Text Full Text PDF PubMed Scopus (2746) Google Scholar, 24Li P. Nijhawan D. Budihardjo I. Srinivasula S. Ahmad M. Alnemri E.S. Wang X. Cell. 1997; 91: 479-489Abstract Full Text Full Text PDF PubMed Scopus (6261) Google Scholar). Apaf-1 directly binds to cytochrome c(24Li P. Nijhawan D. Budihardjo I. Srinivasula S. Ahmad M. Alnemri E.S. Wang X. Cell. 1997; 91: 479-489Abstract Full Text Full Text PDF PubMed Scopus (6261) Google Scholar). To determine whether Nod1 binds to LPS, S100 cell lysates were prepared from HEK293T cells expressing Nod1, and the ability of Nod1 to bind radiolabeled LPS was tested by a modified immunoprecipitation assay. LPS was coimmunoprecipitated with FLAG-tagged Nod1 but not with other FLAG-tagged control proteins (Fig.3 A). Thus, Nod1 is associated with an LPS binding activity present in the cytosolic fraction of HEK293T cells. However, it is possible that Nod1 does not directly bind to LPS and that the association requires other cytosolic factors. For example, dATP or ATP is required for the response of Apaf-1 to cytochrome c (24Li P. Nijhawan D. Budihardjo I. Srinivasula S. Ahmad M. Alnemri E.S. Wang X. Cell. 1997; 91: 479-489Abstract Full Text Full Text PDF PubMed Scopus (6261) Google Scholar). To begin to test this, we first immunoprecipitated Nod1 or IKKβ, as a control protein, with anti-FLAG antibody, and the ability of the immunoprecipitated proteins to bind LPS was tested in nucleotide-free buffer. Immunopurified Nod1 exhibited LPS binding activity, but control IKKβ did not (Fig. 3). These results suggest that Nod1 directly binds LPS. However, we cannot exclude the possibility that Nod1 interacts with LPS through an intrinsic cytosolic factor(s) that is tightly bound to Nod1 and coimmunoprecipitates with Nod1 in the absence of LPS. In plants, theA. thaliana disease-resistant RPS2 gene product that is structurally related to Nod1 and Nod2 can form a protein complexin vivo with the product of the phytopathogenic bacteriumPseudomonas syringae avrRpt2 gene, but the protein complex also contained at least one additional plant protein of ∼75 kDa (16Leister R.T. Katagiri F. Plant J. 2000; 22: 345-354Crossref PubMed Google Scholar). Therefore, further studies are needed to determine whether the association of LPS with Nod1 is direct or mediated through a cytosolic Nod1-interacting protein. In this study, we demonstrate that LPS induces NF-κB activation in HEK293T cell expressing Nod1, whereas parental HEK293T cells are insensitive to LPS. In the human system, the TLR4·MD2·CD14 complex has been demonstrated to serve as a surface receptor for LPS (1Aderem A. Ulevitch R.J. Nature. 2000; 406: 785-787Crossref Scopus (2632) Google Scholar). In addition to the cell surface TLR4 complex, there is mounting evidence that mammalian cells have an intracellular receptor that detects LPS in the cytoplasm of bacteria-infected cells (2Philpott D.J. Yamaoka S. Israel A. Sansonetti P.J. J. Immunol. 2000; 165: 903-914Crossref PubMed Scopus (216) Google Scholar). For example, epithelial cells are unresponsive to extracellular LPS either purified or presented in the context of noninvasive Gram-negative strains (2Philpott D.J. Yamaoka S. Israel A. Sansonetti P.J. J. Immunol. 2000; 165: 903-914Crossref PubMed Scopus (216) Google Scholar). Yet, LPS introduced inside of the epithelial cells activates NF-κB (2Philpott D.J. Yamaoka S. Israel A. Sansonetti P.J. J. Immunol. 2000; 165: 903-914Crossref PubMed Scopus (216) Google Scholar). However, to date, the identification of an intracellular recognition system for LPS and other microbial products remains elusive. Because Nod1 can confer responsiveness to LPS, we propose that Nod1 may act as an intracellular receptor for LPS. Nod1 function might be important in the intracellular response of epithelial cells against invading bacteria, as Nod1 is expressed in intestinal, lung, and nasal epithelial surfaces in the late mouse embryo (11Inohara N. Koseki T. del Peso L. Hu Y. Yee C. Chen S. Carrio R. Merino J. Liu D. Ni J. Núñez G. J. Biol. Chem. 1999; 274: 14560-14568Abstract Full Text Full Text PDF PubMed Scopus (630) Google Scholar). The presence of an intracellular detection system for bacterial LPS would be expected in epithelial surfaces such as those of the gut that are highly exposed to bacteria and bacterial products. In such organs, triggering of an inflammatory response to bacterial products through surface receptors such as TLR4 would be detrimental to the organism. We also demonstrated that HEK293T cells expressing Nod2, another member of the Nod family, also responds to LPS, but Nod1 and Nod2 appear to have different preferences for LPS preparations from different bacteria. These observations suggest that in addition to TLRs, Nod family members may represent another innate immune system for the recognition of a wide array of pathogen products. For example, the genome of the plantA. thaliana contains ∼200 disease-resistant genes encoding intracellular NBD-LRR proteins related to Nod1 and Nod2 (8Dixon M.S. Golstein C. Thomas C.M. van Der Biezen E.A. Jones J.D. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8807-8814Crossref PubMed Scopus (128) Google Scholar). BLAST searches of public data bases revealed that the human genome contains at least 20 Nod1-like genes. 3N. Inohara and G. Nuñez, unpublished data.In future studies, it will be important to elucidate the function and pathogen specificity of mammalian Nod1 family members. We thank Z. Cao, R. Medzhitov, and S. Akira for plasmids, A. Zychlinsky for sBLP, P. Lehmann for mannan, and P. Lucas for critical review of the manuscript.